1. Field of the Invention
The invention relates to enzymes, called oxygenases, which are biologically active proteins that catalyze certain oxidation reactions involving the addition of oxygen to a substance. The transfer of oxygen from an oxygen-donor compound, such as molecular oxygen (O2) and hydrogen peroxide (H2O2), to any of millions of useful aromatic or aliphatic substrate compounds is important in organic chemistry and in many biochemical reactions. Typical oxidation reactions include hydroxylation, epoxidation and sulfoxidation, which are widely used in the production of chemicals including pharmaceuticals and other compounds used in medicine. Enzymes which catalyze or improve oxidation reactions are useful in science and industry. The invention relates to novel oxygenase enzymes having improved properties. The invention also relates to methods of screening for oxygenase enzymes, and more particularly, to methods for identifying oxidation enzymes which exhibit catalytic activity with respect to the insertion of oxygen into aromatic or aliphatic compounds.
The screening method involves introducing an organic substrate compound to an oxygen donor compound in the presence of a test enzyme. Exemplary oxygen donors include molecular oxygen or dioxygen (O2) and peroxides such as hydrogen peroxide (H2O2) and t-butyl peroxide. Exemplary substrates include naphthalene, 3-phenylpropionate, benzene, toluene, benzoic acid, and anthracene. An oxygenated product is formed when the test enzyme has oxidation activity, particularly oxygenase activity, under test conditions.
A coupling enzyme is used to bring together molecules of the oxygenated product into larger molecules or polymers which absorb UV light, produce a color change, or are fluorescent or luminescent. Exemplary coupling enzymes include peroxidases from various microbial and plant sources, such as horseradish peroxidase (HRP), cytochrome c peroxidase, tulip peroxidase, lignin peroxidase, carrot peroxidase, peanut peroxidase, soybean peroxidase, peroxidase Novozyme® 502, as well as laccases such as fungal laccase. The presence and degree of a change in absorbance, color, fluorescence or luminescence can be detected or measured, and indicates the presence of oxygenated product. Detection can be enhanced by a chemiluminescent agent, such as luminol. These techniques provide a reliable indication of oxygenase activity, that is, the production of oxygenated compound by reaction of the oxygen donor with the substrate in the presence of (and mediated by) the enzyme.
The method is preferably carried out in a whole cell environment. A host cell is transformed, using genetic engineering techniques, to express an oxygenase being screened, and may also be engineered to express a coupling enzyme. The method is amenable to large scale screening of enzyme mutants to isolate those with desirable oxygenase activity, for example maximum activity under certain conditions or towards a particular substrate compound. The method is also amenable to screening gene libraries isolated from nature (50).
Oxygenase enzymes typically use molecular oxygen, in the presence of cofactors, coenzymes, and/or ancillary proteins, to add oxygen to a substrate. Oxygen is a highly reactive chemical element. In pure molecular form, it is a gas that is a principal component of air, and is stable as a combination of two oxygen atoms (O2). It appears in water (H2O), in rocks and minerals, in many organic compounds, and is active in many biochemical and physiological processes. Some O2-utilizing enzymes can use other oxygen donors, e.g. peroxides (according to a reaction scheme called the peroxide shunt pathway), but do so poorly, with low activity and a low yield of oxygenated product. Moreover, certain coenzymes, cofactors or ancillary proteins may still be required, although the peroxide shunt does not require the difficult coenzymes, e.g. NAD(P)H, associated with pathways using O2 as a substrate.
The improved oxygenase enzymes of the invention are capable of efficiently catalyzing reactions wherein oxygen is added to a substrate, using oxygen donors other than molecular oxygen, and without requiring certain cofactors, coenzymes, or ancillary redox proteins. These new enzymes have significantly more activity than native enzymes. For example, they are at least twice as active, and typically are ten or more times as active as a wild-type enzyme towards a particular substrate or under particular reaction conditions.
2. Description of Related Art
The publications and reference materials noted here and in the appended Bibliography are each incorporated by reference in their entirety. They are referenced numerically in the text and the Bibliography below.
Catalysts, Enzymes and Oxygenases.
An enzyme is a biological catalyst, typically a protein, which promotes a biochemical reaction. A catalyst enables a chemical reaction to proceed at a faster rate or under different conditions than would otherwise occur. Usually, a catalyst is itself unchanged at the end of the reaction, although oxidative enzymes may be deactivated slowly during these reactions. Oxygenase enzymes that are capable of catalyzing the insertion of oxygen into aromatic (ring-containing) and aliphatic (open-chain) chemical compounds, and other chemical compounds or substrates have many potential applications in pharmaceuticals manufacturing, in the production of chemicals, and also in medicine. Dioxygenases introduce two atoms of oxygen, e.g. both oxygens from a donor such as molecular oxygen (O2). Monooxygenases, also called mixed function oxygenases, add one atom of oxygen to a substrate compound. In these reactions a second oxygen from the oxygen donor may be combined with hydrogen (H+) in a companion reaction, called a reduction reaction, to form water (H2O). Compounds other than molecular oxygen, such as peroxides, can also donate oxygen to a substrate in the presence of various oxygenases.
Common monooxygenation reactions include hydroxylation and epoxidation. In a hydroxylation reaction, oxygen is introduced to a substrate as a hydroxyl group (OH). In an epoxidation reaction, oxygen is introduced as a bridge across two other atoms, typically in place of a double bond between two carbon atoms. This can form an activated or reactive group having a three-member ring of one oxygen atom and two carbon atoms. A common dioxygenation reaction is sulfoxidation. In a sulfoxidation reaction, two oxygen atoms are added to a sulfur atom that is bonded to two other atoms, typically two carbon atoms, each of which is part of a hydrocarbon chain.
The introduction of oxygen to a compound may change its biochemical activity or functionality, and may activate the compound so that it can participate in further chemical reactions. Oxygenated substrates may be used by organisms or industrially, in the synthesis of useful compounds from starting materials or intermediates. Oxygenation may also be useful in the breakdown of compounds, to provide starting materials and intermediates for other reactions. For example, bacteria use oxygenases to digest aromatic compounds.
Problems Addressed by the Invention.
Among the problems addressed by the invention are the significant disadvantages of many known enzyme systems. These problems have prevented commercial use and exploitation of such systems. Many oxygenases, like other enzymes, require expensive coenzymes (e.g. NADPH) and ancillary proteins (e.g. a reductase enzyme), and often must be used in whole cells or reactors with recycled coenzymes, to keep the coenzyme costs low. Known enzymes also are relatively inefficient or unstable under industrial conditions, and may be undesirably deactivated by reaction products or byproducts, or for other reasons. These types of enzyme systems, particularly when used in whole cell reactions, are also prone to competing reactions which can lower the selectivity and yield.
Thus, enzymes which do not require coenzymes, use less coenzymes, or use less expensive coenzymes are desirable. Enzymes which are more efficient, more stable, or which function under different conditions are also desirable. It would also be desirable to provide enzymes which are not adversely affected by competing reactions. Enzymes which promote oxidation of different substrates, which insert oxygen at different positions on a given substrate, insert oxygen more efficiently, or use different oxygen donor compounds would also be desirable, as would enzymes which are more or less specific than known enzymes in catalyzing certain reactions. For example, hydrogen peroxide or other peroxides are good choices of oxidant for fine chemicals manufacturing, as their use would require less specialized equipment, and less cost overall, than molecular oxygen due to the greatly simplified catalyst system. A suitable screening method for oxygenases is also desirable, and would provide an important tool in the discovery and identification of new and improved oxidation enzymes.
Enzymatic oxygenation reactions are particularly intriguing, because directed oxyfunctionalization of unactivated organic substrates remains a largely unresolved challenge to synthetic chemistry. This is especially true for regiospecific reactions, where oxygenation at a specific position of a substrate occurs in only one of two or more possible ways. For example, regiospecific hydroxylation of aromatic compounds by purely chemical methods is notoriously difficult. Reagents for ortho or o-hydroxylation of ring compounds, at positions on the ring which are next or adjacent to each other, are described in the literature. Reagents are also available for para or p-hydroxylation, at positions on the ring which are opposite each other. However, some of these reagents are explosive, and undesirable by-products are usually obtained (1). Likewise, specific oxygenation of enantiomers (mirror-image forms of a compound), is difficult and not well understood. In these reactions, one enantiomer is preferentially oxygenated, but the mirror-image enantiomer of the same compound is poorly oxygenated, or is not oxygenated at all. Similarly, it is difficult to oxygenate a substrate with high enantiospecificty, i.e. so as to create one particular enantiomeric form versus another. Thus, oxygenation to form a particular enantiomer is difficult. Consequently, oxidation enzymes which facilitate particular regiospecific or enantiospecific reactions would be desirable, particularly enzymes which do so under laboratory or industrial conditions, or which do so more efficiently or in some better way.
Oxidation Enzymes.
Various native mono- and dioxygenase enzymes from different microbial, human, plant, and animal sources are known. These include enzymes such as chloroperoxidase (CPO), large numbers of cytochrome P450 enzymes (P450), methane monooxygenases (MMO), toluene monooxygenases, toluene dioxygenases (TDO), biphenyl dioxygenases and naphthalene dioxygenases (NDO). These enzymes have demonstrated the ability to catalyze hydroxylation and many other interesting and useful oxidation reactions. However, they are generally unsuitable for industry due to their inherent complexity, low stability and low productivity under industrial conditions (e.g. in the presence of organic solvents, high concentrations of reactants, etc.).
One class of known oxidation enzymes is the cytochrome P450 enzymes. These heme proteins have iron-containing heme groups and are important monooxygenase enzymes involved in, among other reactions, detoxification of foreign or toxic materials (xenobiotics), drug metabolism, carcinogenesis, and steroid biosynthesis (5 and 6). One exemplary P450 enzyme, P450cam from Pseudomonas putida, whose natural substrate is camphor, is also capable of regiospecific hydroxylation of a variety of substrates including, at a low level of activity, naphthalene (C10H8) a bicyclic aromatic compound (7). However, the catalytic turnover of this enzyme requires the reduced form of nicotinamide-adenine dinucleotide (NADH) as a coenzyme and two ancillary proteins. One of these proteins is putidaredoxin, an iron-sulfur protein (also called a ferredoxin) that acts as an electron carrier to shuttle electrons from NADH. The other ancillary protein is the enzyme putidaredoxin reductase, a flavoprotein which catalyzes the transfer of hydrogen atoms from one substrate to another (8). This requirement for two redox proteins and NADH makes P450cam and other P450 catalysis highly expensive and difficult to use in laboratory and industrial applications. It would be desirable to provide a simpler and more economical P450-type catalyst and hydroxylation system, in particular a system which requires fewer ancillary proteins or coenzymes, or which does not require them at all.
P450 enzymes typically use dioxygen (O2) as the oxygen donor for hydroxylation, adding one oxygen to a substrate compound, such as naphthalene, and forming water with hydrogen and another oxygen as a byproduct. They are most efficient when using dioxygen with expensive coenzymes, such as the reduced forms of nicotinamide-adenine dinucleotide (NADH) or nicotinamide-adenine dinucleotide phosphate (NADPH), collectively “NAD(P)H”. Ancillary proteins may also be needed for efficient enzyme activity. However, various P450s (and, possibly, some MMOs) are able to catalyze the hydroxylation of an organic substrate using a peroxide, such as hydrogen peroxide or alkyl peroxides, via the so-called peroxide shunt pathway (9). Peroxides are compounds, other than molecular O2, in which oxygen atoms are joined to each other. Other oxygen donors include peroxyacids, NaIO4, NaClO2, and iodosyl benzene.
Nordblom et al.
(11) studied hydroperoxide-dependent substrate hydroxylation by liver microsomal P450 in hepatic microsomes. A variety of substrates were shown to be attacked by the enzyme in the presence of cumene hydroperoxide. Using benzphetamine as the substrate, it was also shown that other peroxides, including hydrogen peroxide, peracids and sodium chlorite, could be used in place of oxygen (11). Rahimtula et al. (12) showed that cumene hydroperoxide is capable of supporting the hydroxylation of various aromatic compounds (biphenyl, benzpyrene, coumarin, aniline) by cytochrome P450 in hepatic microsomes. Unfortunately, native cytochrome P450 is rapidly deactivated by peroxides and other oxidants.
The enzyme chloroperoxidase (CPO) from Caldariomyces fumago has an active site whose structure is similar to cytochrome P450 enzymes. CPO will catalyze various oxidation reactions, including enantioselective hydroxylation, epoxidation and sulfoxidation, using peroxides. This enzyme utilizes peroxide efficiently but cannot utilize molecular oxygen because it does not have the coenzyme machinery of the P450 enzymes. CPO also provides an example of an enzyme that is deactivated by reactive intermediates. Heme alkylation by the epoxide product in the CPO-catalyzed epoxidation of 1-alkenes results in CPO deactivation.
Heme oxygenases such as P450s and heme peroxidases, which are peroxidase enzymes that contain the heme prosthetic group, are generally prone to deactivation via oxidation of the porphyrin ring in the heme substrate, by reaction with so-called suicide inhibitors formed during catalysis, and also by formation of Compound III (for peroxidases). Compound III is an intermediate enzyme-substrate-oxygen-iron complex, sometimes referred to an oxyperoxidase. For example, the enzyme horseradish peroxidase (HRP) is deactivated during the oxidation of phenol compounds, e.g. six-member hydrocarbon ring structures containing one or more hydroxyl (OH) groups. In theory, this may be due to the formation of phenoxy radicals which react with oxygen to form a reactive peroxy-radical species. Compound III forms in the presence of excess hydrogen peroxide and is not involved in the reaction cycle. However, its accumulation reduces the amount of active enzyme. Compound III stability in turn depends on the specific enzyme.
The rates of all of these deactivation pathways depend on the protein framework, i.e. the particular proteins, structures and conditions involved. They all are therefore amenable to improvement by mutations. This includes oxygenases that are more suitable to function in the presence of high concentrations of hydrogen:peroxide, or other peroxides or oxygen-donating agents. Improved oxygenases also include those which are more resistant to deactivation, do not require coenzymes or use them more efficiently, function under different conditions or with different specificities, or which hydroxylate different substrates or a variety of substrates, or which do so more efficiently. As one example, it would be desirable to make modified P450 enzymes that are functionally similar or equivalent to CPO, or which share desirable features of CPO. An improved P450 enzyme of this kind, for example, would have the ability to oxygenate a substrate or substrates using a peroxide, e.g. hydrogen peroxide, without expensive coenzymes, and with a high efficiency and improved resistance to deactivation.
Enzyme Modification.
The observed constraints on the use of native enzymes are thought to be a consequence of evolution. Enzymes have evolved in the context and environment of a living organism, to carry out specific biological functions under conditions conducive to life—not laboratory or industrial conditions. In some cases, evolution may favor or even require less than optimally efficient enzymes. For example, detoxication enzymes, such as cytochrome P450 enzymes, function to help convert foreign (xenobiotic) chemical compounds into other compounds that an organism can use, that are not toxic, or that are present in non-toxic amounts. In order to deal with environmental conditions or foreign compounds an organism has not encountered before, detoxification enzymes may attack a relatively large number of substrates, and may accidentally produce products that are as or more toxic than the substrate. Thus, maximizing the flow of potentially harmful foreign substrates for processing, e.g. using an overly efficient catalyst, may not be the best evolutionary strategy. This is particularly true when there is a time-dependent xenobiotic profile, meaning that the organism can only safely handle so much foreign material at a time (2). In this situation, a less than maximally active enzyme that is appropriately balanced to the particular needs of the organism and its environment would be a better evolutionary goal. In a laboratory or industrial setting, it is desirable to provide enzymes which are more active, and process more substrate more rapidly.
Thus, the output, efficiency, working conditions, stability and other properties of known enzymes are not thought to be unalterable, nor are they limitations which are seen as intrinsic to the nature of these catalysts as proteins. It is possible that these native catalysts can be evolved in vitro, or that analogous catalysts can be otherwise developed, to alter or enhance the enzyme's properties, for example to obtain much more efficient laboratory or industrial oxidative catalysts. Enzyme selectivity and substrate specificity may also be altered to better match the needs of the synthetic chemist. Improved catalysts can also be obtained by screening cultures of native organisms or expressed gene libraries (3).
One technique which may be applied to the discovery of improved catalytic enzymes is directed evolution. Directed evolution is a procedure by which the evolutionary process is accelerated in vitro to produce mutant enzymes which have certain desired characteristics. An example of the use of directed evolution for identifying and isolating improved para-nitrobenzyl esterases is set forth in U.S. Pat. No. 5,741,691. See also, U.S. Pat. No. 5,811,238 (13). Other techniques, such as random mutagenesis, may also be used to obtain new enzymes. Improved enzymes may also be discovered in nature.
According to a preferred embodiment of the invention, directed evolution or random mutagenesis can be used to produce an array of efficient catalysts which can perform oxidations using agents other than dioxygen (O2) as the oxidant. For example, peroxides such as hydrogen peroxide (H2O2) may be used. Directed evolution can also be used to alter the properties of oxidative enzymes that use molecular oxygen. A variety of such enzymes, including cytochrome P450s, other monooxygenases, and dioxygenases such as toluene dioxygenase, facilitate useful oxygenation reactions. It is desirable to alter the reactivities, selectivities and stabilities of these enzymes to produce improved enzymes. An important tool for finding improved oxidation biocatalysts in nature, by directed evolution, by random mutagenesis, or by other means, is a sensitive, accurate and rapid screening method. Accordingly, there is a need to develop new and improved screening methods for enzymes which function as oxygenases. In particular there is a need for screening methods which are well-suited for use in connection with directed evolution procedures.